Expression of hepatitis C virus non‐structural 5A protein in the liver of transgenic mice

Abstract

Hepatitis C virus (HCV) is a major etiologic agent for chronic hepatitis worldwide often leading to the development of cirrhosis and hepatocellular carcinoma. However, the mechanism for development of chronic hepatitis or hepatocarcinogenesis by HCV remains unclear. HCV NS5A protein possesses many intriguing properties, including sequestration of p53 in the cytoplasm, downregulation of p21 protein, activation of STAT3, and inhibition of tumor necrosis factor-alpha-mediated apoptosis. Thus, we investigated whether this viral protein has oncogenic property in vivo. In the absence of an efficient cell culture system for virus growth and a suitable small animal model for HCV infection, transgenic FVB mice were generated by targeting the HCV NS5A genomic region cloned under the control of a liver-specific apoE promoter or mouse major urinary promoter (MUP). The apoE promoter is constitutively expressed in liver, on the other hand, the MUP is developmentally regulated and expressed in the liver after birth. Reverse transcription polymerase chain reaction and Western blot analysis indicated establishment of HCV NS5A transgene expression in several lines from both groups of mice. Immunohistochemical studies suggested the presence of NS5A in the cytoplasm of hepatocytes. The transgenic animals were phenotypically similar to their normal littermates and did not exhibit a major histological change within the liver up to 24 months of age. Our results suggested HCV NS5A protein is not directly cytopathic or oncogenic in this FVB transgenic mouse model, although this viral protein promotes cell growth in vitro. These animals will be a valuable model of HCV immunopathology as well as for evaluation of siRNA, interferon and other cytokine therapies.

Toll-like receptor 4 mediates synergism between alcohol and HCV in hepatic oncogenesis involving stem cell marker Nanog

“…However, whether NS5A induces TLR4 in the liver of the whole animal is yet to be determined. To address this question, NS5A was expressed in a hepatocyte-specific manner in mice with the use of an apoE promoter (23,24). RT-PCR and immunoblot analyses confirmed induction of TLR4 mRNA and protein levels in the NS5A Tg mouse liver compared with the wild-type (WT) mouse liver ( Fig.…”

Signal transduction cascades and hepatitis B and C related hepatocellular carcinoma

“…135 However, other forms (p50, p53) have also been described in the Huh-7 hepatoma cell line. 136 Though expression of this protein in transgenic mice has not demonstrated direct oncongenicity, 137,138 NS5a has been associated with cell transformation in vitro. [139][140][141] Transfecting murine embryo fibroblasts (NIH 3T3) with NS5a results in a transformed phenotype, defined by increased proliferation, colony formation in soft-agar medium, and tumor formation in nude mice.…”

Expression of hepatitis C virus nonstructural 4B in transgenic mice

“…Among the nonstructural proteins, NS5A and NS4B are the primary candidates, but their functions are largely unknown. It has been reported that transgenic mice expressing NS5A protein did not show any pathological changes (Majumder et al, 2003), which indicates that NS5A protein alone is not cytopathic or oncogenic to hepatocytes. In this study, we first screened for possible pathological functions, which we inferred, of the NS4B protein in vivo.…”

“…An understanding of HCV-mediated pathogenesis and the control of HCV infection have been impaired in part because of the unavailability of an efficient cell culture system for virus growth and a convenient small animal model. Advances in the field of transgenic technology have offered us unique opportunities to develop valuable rodent models for human diseases (Majumder et al, 2003). Currently published data suggest that the expression of the HCV virus gene from the HCV genotype 1b (Japanese strains) in the genetic background of the C57BL/6J mouse can induce hepatic alterations (Moriya et al, 1997;Moriya et al, 1998;Lerat et al, 2002).…”

“…Currently published data suggest that the expression of the HCV virus gene from the HCV genotype 1b (Japanese strains) in the genetic background of the C57BL/6J mouse can induce hepatic alterations (Moriya et al, 1997;Moriya et al, 1998;Lerat et al, 2002). However, transgenic mice expressing the HCV gene that were established using another genotype of HCV or another genetic background of mouse strain (Kawamura et al, 1997;Pasquinelli et al, 1997;Majumder et al, 2003;Wang et al, 2004) failed to generate hepatic alterations. In Japan, approximately 15, 90, and 80% of patients with sporadic acute hepatitis, post-blood transfusion chronic hepatitis, and HCC, respectively, are positive for HCV infection (Watashi et al, 2003).…”